P1468: PIEZO1 GAIN-OF-FUNCTION VARIANTS ACCOUNT FOR STRESSED ERYTHROPOIESIS BY ALTERING APOPTOSIS, HYPOXIA CELL RESPONSE, AND GLYCOLYSIS
Authors: Barbara Eleni Rosato, Vanessa D’onofrio, Antonella Nostroso, Roberta Marra, Federica Maria Esposito, Maddalena Raia, Giuseppe D’alterio, Mario Capasso, Achille Iolascon, Roberta Russo, Immacolata Andolfo
Journal: HemaSphere (2023)

Topic: 28. Enzymopathies, membranopathies and other anemias Background: PIEZO1 is a mechanosensitive cation channel involved in various physiological processes as a sensor of mechanical forces. In erythrocytes, it is involved in the regulation of hydration and cellular volume. Gain-of-function (GoF) mutations slow the inactivation kinetics of the channel leading to dehydrated hereditary stomatocytosis (DHS). Despite the variable clinical presentation, most patients present mild to severe hemolytic anemia, increased hemolysis markers, and iron overload. Analysis of erythroid progenitor cells of DHS patients showed a delayed erythroid differentiation, mutations dependent. Besides, the constitutive GOF Piezo1 mice showed stressed erythropoiesis with increased erythrocytes turnover. Aims: To dissect the role of PIEZO1 during stressed erythropoiesis in DHS to find new druggable targets. Methods: ELISA assay, in-vitro differentiation of immortalized erythroid progenitors (Hudep2) (with erythroid differentiation medium) and engineered for PIEZO1 R2456H (Hudep2-KI) variant; flow cytometry, RNA-sequencing. Results: We herein analyzed 80 DHS patients by measuring BMRI [(absolute reticulocyte count) × (patient’ Hb/normal Hb)], EPO, and ERFE levels. We observed mutation-dependent dyserythropoietic features similar to those measured in CDAII patients (n=55, characterized by dyserythropoiesis). Bone marrow examination confirmed these features, showing erythroid hyperplasia and double nuclei in erythroid lineage. Our data support the presence of dyserythropoiesis in DHS patients depending on the specific mutation. The engineered erythroid model of DHS, Hudep2-KI, was induced to erythroid differentiation for 12 days and compared to Hudep2-WT. At steady state and during differentiation, no significant changes were observed in earlier differentiation markers expression, CD36 and CD49d. A significant reduction of CD235a expression was measured at last day of differentiation, confirming that PIEZO1-mediated alteration occurs at the latest stages of differentiation. The morphological analysis of differentiating cells showed, from day 7, that KI cells strongly reduced their proliferation rate (1.5-fold increase vs day 0) compared to Hudep2-WT (3.1-fold increase vs day 0). At 12 days of differentiation, KI cells presented an halved cell count compared to WT, with a strong reduction of orthochromatic normoblasts percentage (13.7%) compared to WT (54.5%). This was paralleled with a relative increase of reticulocytes (Hudep2-WT: 45.5% vs Hudep2-KI: 86.3%). To identify the altered signaling pathways involved in the deregulation of differentiation, RNAseq was performed during differentiation. Firstly, we selected the significantly deregulated genes in KI cells compared to WT for each time point and a gene ontology analysis was carried out. We observed that most genes were significantly deregulated at day 10. The most enriched biological processes, through the three datasets, were related to the apoptotic process, cellular response to hypoxia, and glycolytic process. Summary/Conclusion: Herein we demonstrated that PIEZO1 GOF mutation has a role in stressed erythropoiesis in both patients and the erythroid cellular system. PIEZO1 GOF variants, by increasing the intracellular calcium concentration, affect the latest stages of differentiation through the deregulation of several pathways as apoptosis, response to hypoxia, and glycolysis. The data are under confirmation in CD34+ cells from DHS patients, and in PIEZO1 GOF mice and will be compared to metabolomic data. The identified deregulated markers will be validated as possible druggable targets for dyserythropoiesis. Keywords: Erythroid differentiation, Erythropoieisis, Hemolytic anemia, RNA-seq